Epileptic activity triggers rapid ROCK1-dependent astrocyte morphology changes.

Long-term modifications of astrocyte function and morphology are well known to occur in epilepsy. They are implicated in the development and manifestation of the disease, but the relevant mechanisms and their pathophysiological role are not firmly established. For instance, it is unclear how quickly the onset of epileptic activity triggers astrocyte morphology changes and what the relevant molecular signals are. We therefore used two-photon excitation fluorescence microscopy to monitor astrocyte morphology in parallel to the induction of epileptiform activity. We uncovered astrocyte morphology changes within 10-20 min under various experimental conditions in acute hippocampal slices. In vivo, induction of status epilepticus resulted in similarly altered astrocyte morphology within 30 min. Further analysis in vitro revealed a persistent volume reduction of peripheral astrocyte processes triggered by induction of epileptiform activity. In addition, an impaired diffusion within astrocytes and within the astrocyte network was observed, which most likely is a direct consequence of the astrocyte remodeling. These astrocyte morphology changes were prevented by inhibition of the Rho GTPase RhoA and of the Rho-associated kinase (ROCK). Selective deletion of ROCK1 but not ROCK2 from astrocytes also prevented the morphology change after induction of epileptiform activity and reduced epileptiform activity. Together these observations reveal that epileptic activity triggers a rapid ROCK1-dependent astrocyte morphology change, which is mechanistically linked to the strength of epileptiform activity. This suggests that astrocytic ROCK1 signaling is a maladaptive response of astrocytes to the onset of epileptic activity.

Heterogeneity and Development of Fine Astrocyte Morphology Captured by Diffraction-Limited Microscopy.

The fine processes of single astrocytes can contact many thousands of synapses whose function they can modulate through bi-directional signaling. The spatial arrangement of astrocytic processes and neuronal structures is relevant for such interactions and for the support of neuronal signaling by astrocytes. At the same time, the geometry of perisynaptic astrocyte processes is variable and dynamically regulated. Studying these fine astrocyte processes represents a technical challenge, because many of them cannot be fully resolved by diffraction-limited microscopy. Therefore, we have established two indirect parameters of astrocyte morphology, which, while not fully resolving local geometry by design, provide statistical measures of astrocyte morphology: the fraction of tissue volume that astrocytes occupy and the density of resolvable astrocytic processes. Both are straightforward to obtain using widely available microscopy techniques. We here present the approach and demonstrate its robustness across various experimental conditions using mainly two-photon excitation fluorescence microscopy in acute slices and in vivo as well as modeling. Using these indirect measures allowed us to analyze the morphology of relatively large populations of astrocytes. Doing so we captured the heterogeneity of astrocytes within and between the layers of the hippocampal CA1 region and the developmental profile of astrocyte morphology. This demonstrates that volume fraction (VF) and segment density are useful parameters for describing the structure of astrocytes. They are also suitable for online monitoring of astrocyte morphology with widely available microscopy techniques.

LTP Induction Boosts Glutamate Spillover by Driving Withdrawal of Perisynaptic Astroglia.

Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.

Central connections of the trigeminal motor command system in the weakly electric Elephantnose fish (Gnathonemus petersii).

The highly mobile chin appendage of Gnathonemus petersii, the Schnauzenorgan, is used to actively probe the environment and is known to be a fovea of the electrosensory system. It receives an important innervation from both the trigeminal sensory and motor systems. However, little is known about the premotor control pathways that coordinate the movements of the Schnauzenorgan, or about central pathways originating from the trigeminal motor nucleus. The present study focuses on the central connections of the trigeminal motor system to elucidate premotor centers controlling Schnauzenorgan movements, with particular interest in the possible connections between the electrosensory and trigeminal systems. Neurotracer injections into the trigeminal motor nucleus revealed bilateral, reciprocal connections between the two trigeminal motor nuclei and between the trigeminal sensory and motor nuclei by bilateral labeling of cells and terminals. Prominent afferent input to the trigeminal motor nucleus originates from the nucleus lateralis valvulae, the nucleus dorsalis mesencephali, the cerebellar corpus C1, the reticular formation, and the Raphe nuclei. Retrogradely labeled cells were also observed in the central pretectal nucleus, the dorsal anterior pretectal nucleus, the tectum, the ventroposterior nucleus of the torus semicircularis, the gustatory sensory and motor nuclei, and in the hypothalamus. Labeled terminals, but not cell bodies, were observed in the nucleus lateralis valvulae and the reticular formation. No direct connections were found between the electrosensory system and the V motor nucleus but the central connections identified would provide several multisynaptic pathways linking these two systems, including possible efference copy and corollary discharge mechanisms.

Control of astrocyte morphology by Rho GTPases.

Astrocytes modulate and support neuronal and synapse function via numerous mechanisms that often rely on diffusion of signalling molecules, ions or metabolites through extracellular space. As a consequence, the spatial arrangement and the distance between astrocyte processes and neuronal structures are of functional importance. Likewise, changes of astrocyte structure will affect the ability of astrocytes to interact with neurons. In contrast to neurons, where rapid morphology changes are critically involved in many aspects of physiological brain function, a role of astrocyte restructuring in brain physiology is only beginning to emerge. In neurons, small GTPases of the Rho family are powerful initiators and modulators of structural changes. Less is known about the functional significance of these signalling molecules in astrocytes. Here, we review recent experimental evidence for the role of RhoA, Cdc42 and Rac1 in controlling dynamic astrocyte morphology as well as experimental tools and analytical approaches for studying astrocyte morphology changes.

Spatial properties of astrocyte gap junction coupling in the rat hippocampus.

Gap junction coupling enables astrocytes to form large networks. Its strength determines how easily a signalling molecule diffuses through the network and how far a locally initiated signal can spread. Changes of coupling strength are well-documented during development and in response to various stimuli. Precise quantification of coupling is needed for studying such modifications and their functional consequences. We therefore explored spatial properties of astrocyte coupling in a model simulating dye loading of single astrocytes. Dye spread into the astrocyte network could be characterized by a coupling length constant and coupling anisotropy. In experiments, the fluorescent marker Alexa Fluor 594 was used to measure these parameters in CA1 and dentate gyrus of the rat hippocampus. Coupling did not differ between regions but showed a temperature-dependence, partially owing to changes of intracellular diffusivity, detected by measuring coupling length constants but not the more variable cell counts of dye-coupled astrocytes. We further found that coupling is anisotropic depending on distance to the pyramidal cell layer, which correlated with regional differences of astrocyte morphology. This demonstrates that applying these new analytical approaches provides useful quantitative information on gap junction coupling and its heterogeneity.

Neuronal adaptation involves rapid expansion of the action potential initiation site.

Action potential (AP) generation is the key to information-processing in the brain. Although APs are normally initiated in the axonal initial segment, developmental adaptation or prolonged network activity may alter the initiation site geometry thus affecting cell excitability. Here we find that hippocampal dentate granule cells adapt their spiking threshold to the kinetics of the ongoing dendrosomatic excitatory input by expanding the AP-initiation area away from the soma while also decelerating local axonal spikes. Dual-patch soma-axon recordings combined with axonal Na(+) and Ca(2+) imaging and biophysical modelling show that the underlying mechanism involves distance-dependent inactivation of axonal Na(+) channels due to somatic depolarization propagating into the axon. Thus, the ensuing changes in the AP-initiation zone and local AP propagation could provide activity-dependent control of cell excitability and spiking on a relatively rapid timescale.